Novel Mechanisms for Coreceptor Switching

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DESCRIPTION (provided by applicant): Entry of HIV-1 into target cells is mediated by binding of CD4 and CCR5, but a substantial fraction of infected individuals have sequence changes in the envelope gene that add or replace entry via CCR5 with entry via CXCR4, a process termed "coreceptor switching." The proposed experiments will test several novel hypotheses to explain how coreceptor switching takes place, and why a history of antiviral therapy and low CD4 T cell counts increases coreceptor switching. One hypothesis is that very rapid sequence changes are introduced by recombination events between contemporaneous CCR5 (R5) viruses and CXCR4 (X4) sequences recovered from latently-infected, long-lived memory cells. A competing hypothesis is that bursts of mutation and positive selection drive rapid sequence changes. These will be distinguished by determining the incidence of recombinant envelope genes in entry competent molecular clones from a cohort of 49 patients with documented coreceptor switching events. In those individuals with recombination events that influence coreceptor use, new envelope genes will be cloned by activating the resting memory T cell pool recovered from samples of patient cells stored prior to coreceptor switching. The nucleotide sequences of these genes will be compared to those that contributed to later coreceptor switching to determine if they were the origin of the recombination events. Prior antiviral therapy with reverse transcriptase inhibitors may lead to a higher mutation rate that differently impacts R5 viruses. Neutral, synonymous mutations should accumulate in viruses from subjects with RT inhibitor resistance. Very low CD4 T cell counts leads to a higher proportion of infected cells, which may favor dual infection and recombination. The impact of prior therapy and nadir CD4 T cell counts on events leading to coreceptor switching will be determined by longitudinal studies of virus samples before and after switching. These experiments will lead to a better understanding of how coreceptor switching takes place, and provide new insights into the potential consequences of activating latent infection to eradicate HIV-1.